Parvovirus designates a genus of the virus family Parvoviridae. The parvovirus genus comprises a number of small, icosaedric viruses that can replicate in the absence of a helper virus. Parvovirus contains a single-stranded DNA having a length of about 5.000 bp. At the 3′ and 5′ ends of the DNA there is one palindromic sequence each. The DNA codes for two capsid proteins, VP1 and VP2, as well as for two regulatory non-structure proteins, NS1 and NS2. The latter proteins are phosphorylated and show nuclear or both cytoplasmic and nuclear localization, respectively. The two capsid proteins VP1 and VP2 are encoded by overlapping open reading frames so that the VP2 encoding region is entirely comprised within the VP1 encoding region. In a natural infection capsid proteins are expressed in a VP1:VP2 ratio of 1:10 due to alternative splicing. Recently it has been shown that the region of the capsid proteins which is specific for VP1 (N-terminal region) contains motifs common to cellular phospholipase A and indeed exerts this activity in vitro. The calcium dependent, secreted PLA2 to which parvovirus VP1 shares similarities, takes part in signaling pathways that involve cell lysis by permeabilizing membranes. On the other hand, NS1 has been shown to be regulated by members of the protein kinase C family, which in turn are subject to regulation through phospholipases.
Parvoviruses are usually well-tolerated by populations of their natural host, in which they persist without apparent pathological signs. This is due to both the protection of foetuses and neonates by maternal immunity, and the striking restriction of parvovirus replication to a narrow range of target proliferating tissues in adult animals. This host tolerance concerns especially rodent parvoviruses, for example the minute virus of mice (MVM) and H-1 virus in their respective natural hosts, namely mice and rats. In addition, humans can be infected with the latter viruses, without any evidence of associated deleterious effects from existing epidemiological studies and clinical trials. On the other hand, autonomous parvoviruses have been shown to preferentially propagate in and to kill neoplastically transformed cells. In addition, they consist of a class of viruses that, despite causing viremia in their infected hosts, mostly produce an apathogenic infection. For these reasons, autonomous parvoviruses are thought to be excellent tools for cancer gene therapy. Particular interests are focused on recombinant vectors maintaining their natural oncotropism, as well as their oncolytic and oncosuppressive potential.
NS1, the major non-structural protein is necessary for viral DNA replication and participates in the regulation of viral gene expression. Particularly, NS1 transactivates the promoter P38 and exhibits DNA-binding, helicase and DNA-nicking activities. Furthermore, NS1 induces cytotoxic and/or cytostatic stress in sensitive host cells.
Therefore, it seems advisable to keep NS1 within parvoviral vectors, in order to (a) maintain their specific competence for NS1-dependent DNA replication and gene expression in tumour cells, resulting in an enhanced production of therapeutics agents at the desired locus (oncotropism), (b) take advantage of NS1 cytotoxicity to kill neoplastic cells and shed tumour antigens to elicit anti-cancer immunity (oncolysis), and (c) achieve still unknown effects that may contribute to the persistence (memory) of a tumour-free status (oncosuppression).
Current parvoviral vectors harbor the wild type genes (for the above discussed reasons) and either of a variety of different transgenes aimed to induce anti-tumoral responses. In most applications considered to date, the transgenes were chosen for their capacity to upmodulate the host cellular immunity against cancer. While the transgene expression induced by such vectors was successfully targeted at the location of the tumour and was especially high, duration of the therapy was relatively short, i.e. three to five days, which is likely due to the NS1-induced killing of transduced cells. New developments concerning the regulation of the NS1 protein have shown that the cytotoxicity of this protein is a regulated process. Furthermore, NS1 variants have been produced by site-directed mutagenesis of putative regulatory elements, which are up- and down-modulated regarding their cytotoxic potential while keeping replicative functions (European patent application No. 99115161.4). These variants may allow a prolongated expression of transgenes harbored by parvoviral vectors (less toxic variants) or endow infectious parvoviruses with an enhanced oncotoxicity (more toxic variants).
Considering the use of NS1 as an oncotoxin in the context of parvoviral, but also non-parvoviral vectors it should be stated that, though able to induce apoptosis in certain cancer cells (leukemias), NS1 alone often causes only a cytostatic effect that is accompanied by morphological changes (cell shrinking) and a dramatic disorganisation of the cytoskeleton in the absence of cell lysis or apoptotic signs. NS1-expressing cells remain on a long-term in this state that has, thus, common features with a terminal differentiation. This NS1-mediated induction of a cellular terminal state is interesting on its own right for limiting tumour cell proliferation, yet it is not suitable to trigger the uptake of tumour-associated antigens (TAAs) by presenting cells of the immune system, which requires tumour cell death. This is a very serious limitation of the use of NS1 alone: Given that only a minor fraction of tumour cells can be expected to be killed, it is crucial that the direct toxicity of the vector (affecting only transduced cells) is enhanced by the immune system in order to achieve a bystander suppressive effect on non-infected tumour cells.